This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are of bacterial origin, i.e., isolated from Salmonella typhimurium. In particular, the invention relates to isolated nucleic acid molecules, such as DNA and RNA encoding a uracil transport protein and a uracil phosphoribosyltransferase and uses thereof.
In most organisms, the biosynthesis of the purine, pyrimidine, and pyridine nucleotides, as well as the aromatic amino acids, histidine, and tryptophan, involves a group of ten enzymes known as phosphoribosyltransferases (PRTases). Each of these enzymes is highly specific for a nitrogenous base, generally aromatic, a divalent metal ion and α-D-5-phosphoribosyl 1-pyrophosphate (PRPP). In all cases, cleavage of the pyrophosphate moiety of PRPP is accompanied by the anomeric inversion of the ribofuranose ring resulting in a β-N riboside monophosphate. In vertebrates, several PRTases exhibit a striking organ specificity while others are found in varying levels in most tissues. In all organisms, the PRTases are subcellularly confined to the soluble cytoplasmic fractions. In mammals, a single enzyme, orotate phosphoribosyltransferase (OPRTase), is responsible for the salvage of pyrimidine bases. However, in bacteria, yeast, and plant cells a uracil-specific enzyme is also found—Uracil phosphoribosyltransferase (Uracil phosphoribosyltransferase)
Uracil phosphoribosyltransferase of bacterial origin catalyzes the conversion of uracil and 5-phosphoribosyl α-1-pyroposphate (PRib-PP) to uridine-5′-monophosphate (UMP) and PPi. See Neuhard et al., Metabolism oh Nucleotides, Nucleosides and Nucleobases in Microorganisms (Munich-Petersen A., ed.) Academic Press, New York, 95–148. Importantly, the bacterial uracil phosphoribosyltransferase, although absent in mammalian cells is nevertheless functionally equivalent to orotate phosphoribosyltransferase or uridine-5′-monophosphate synthase of mammalian cells and has a fundamental importance in the utilization of endogenous uracil formed by degradation of pyrimidine and in the utilization of exogenous uracil, cytosine and uridine for pyrimidine synthesis. This has been demonstrated in Saccharomyces cerevisiae, infra.
The UPP gene from Escherichia coli encodes for the enzyme uracil phosphoribosyltransferase and has been isolated by Anderson et al., Eur J. Biochem, 204: 51–56 (1992). (Andersen et al., 1992). Mutants of Escherichia coli lacking the enzyme uracil phosphoribosyltransferase but with an intact uracil transport system fail to grow on uracil as a pyrimidine source and they excrete uracil into the culture medium. See Malloy A., et al. FEBS Letts., 5: 211–213 (1969). Furthermore they are resistant to 20 μM 5-fluorouracil, this being a phenotype which has been used in the selection of UPP mutants.
The role of uracil phosphoribosyltransferase in the salvage of endogenously formed uracil and in the utilization of exogenous uracil and cytosine has been demonstrated in several microorganisms including Escherichia coli. The pyrimidine salvage enzymes enable the cells to utilize preformed nucleobases and nucleosides either from the growth medium or from degradation products of cellular nucleic acids.
The nucleotide sequence of the gene encoding URPTase from Saccharomyces cerevisiae has recently been published. See Kern, et al., Gene, 88: 149–157 (1990). This gene encodes a 28.7-kDa protein. The deduced amino acid sequences of the Uracil phosphoribosyltransferase from Escherichia coli and Saccharomyces cerevisiae have been compared and found to share some similarities in discrete areas despite only 32% overall identities.
Natural nucleosides and nucleobases are important metabolites and have a myriad of physiological effects in many organs, systems and species. As a result of the varied metabolic fates of nucleosides and nucleobases and their key role in nucleic acid metabolism, many analogues of these compounds have been synthesized over the past 40 years with the aim of developing clinically useful drugs. Synthetic nucleosides have important applications in chemotherapy of the leukemias and as antiviral agents, e.g. cytosine arabinoside (araC), acyclovir, azidothymidine, 5-fluorodeoxyuridine and 5-fluorouracil. New analogues of nucleobases and nucleosides acting as antimetabolites and antibiotics continue to be synthesized and evaluated for prospective therapeutic application. Some nucleosides and nucleobases can reverse the effects of particular inhibitors of de novo pyrimidine and purine synthesis such as methotrexate, 5-fluorouracil and N-phosphonoacetyl-L-aspartate.
Bacterial uracil phosphoribosyltransferase is functionally equivalent to orotate phosphoribosyltransferase or uridine-5′-monophosphate synthase of mammalian cells. These enzymes mediate the conversion of 5-fluorouracil (5-FU)1, to 5-fluorouridine 5′ monophosphate (5-FUMP). 5-fluorouridine 5′ monophosphate is subsequently converted 5-FdUDP and 5-FdUMP in the mammalian de novo pyrimidine pathway. Each 5-FdUNT is an irreversible inhibitor of thymidylate synthase (Thy-A) and results in dTIP starvation and subsequent apoptosis. This conversion is one of the requisite pathways to achieve cytotoxic effects of 5-fluorouracil. See Kawamura, K et al., Cancer Gene Ther., 7: 637–43 (2000) whose data corroborate the above conclusions regarding the ability of bacterially derived uracil phosphoribosyltransferase to convert 5-fluorouracil to an active metabolite 5-fluorouridine-5′-monphosphate as does mammalian orotate phosphoribosyltransferase. It has been suggested that the bacterial uracil phosphoribosyltransferase encoding gene that is absent in mammalian cells, when expressed in tumor cells, can effectively enhances the cytotoxic effect attending 5-fluorouracil in the transduced cells. 1 5-FU has been approved by the FDA for the treatment of cancer. However, it is relatively toxic to patients. As such, its dose must be minimized to avoid adverse reactions. See Pinedo, et al., J. Clin. Oncol., 6: 1653–1664 (1988).
The data suggest that uracil phosphoribosyltransferase gene therapy with 5-fluorouracil can sensitize the antitumor effect of 5-fluorouracil. Consequently, this approach is a new chemosensitizing strategy for cancer gene therapy and a more feasible modality for the treatment of bladder cancer.
Researchers have also reported that infecting human colon cancer cells with an adenovirus carrying the Escherichia coli gene for uracil phosphoribosyltransferase makes them much more sensitive to treatment with 5-fluorouracil. The data demonstrate that the adenoviral-mediated transfer of the Escherichia col UPP gene enhances both the DNA- and RNA-directed activating anabolisms of 5-fluorouracil resulting in sensitizing human colon cancer cells to treatment with 5-FU, thus suggesting that “the UPRT/5-fluorouracil system can be regarded as a new biochemical modulation of fluorouracil therapy for colorectal cancer treatment.” See Koyama, F et al., Eur J Cancer, 36:2403–2410 (2000).
As well, Sunamura, M. et al., Nippon Rinsho, 59: 98–103 (2001) report that the transfection of a bacterial UPP gene into pancreatic cells resulted in a significant change in the sensitivity of pancreatic cells against 5-fluorouracil. See also Adachi, Y. et al., Hum. Gene Ther., 11 :77–89 (2000). Similar results have been reported by Inaba M et al., Jpn J Cancer Res. 90: 349–354 (1999) in a human stomach cancer cell line. As well, Kanai et al., Cancer Res, 58: 1946–51 (1998) report that adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase encoding gene resulted in a marked sensitization of colon, gastric, and pancreatic cancer cell lines. More, 5-fluorouracil treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with a bacterial uracil phosphoribosyltransferase encoding gene resulted in a significant in vivo antitumor effect.
Nucleoside and nucleobase transportation is common in a large variety of organisms and has many different physiological effects (Griffith and Jarvis 1996). Physiological nucleosides and nucleobases, and most nucleoside analogues, are hydrophilic, and specialized transport systems are required for their movement into or out of cells. The presence or absence of nucleoside and nucleobase transporters in cells and organisms will have an important impact on the pharmacokinetics, and the disposition and in vivo biological activity of physiological occurring compounds as well as nucleoside and nucleobase drugs.
Several references describe that the uracil transport protein is necessary for uracil uptake at low exogenous uracil concentrations, even under conditions with high uracil phosphoribosyltransferase activity. Investigators have suggested that uracil enters the cytoplasm by facilitated diffusion across the cytoplasmic membrane where the uracil transport protein is a membrane-bound facilitator.
It is noteworthy that none of the prior art references describe the isolation of a uracil transport protein or the gene encoding this protein—uraA or a UPP gene encoding for uracil phosphoribosyltransferase from Salmonella typhimurium. 
As such, the availability of the disclosed isolated nucleic acid molecules that will fulfill the above referenced voids in the prior art and will provide detailed information of the encoded proteins' structure and function based on predictions drawn from other sources.
In addition, the availability of the disclosed isolated nucleic acid molecules will allow for improving the therapeutic efficacy of current cancer treatment protocols as well as allowing for the development of therapeutic candidates that are capable of sensitizing cancerous cells to treatment with conventional anti-tumor drugs etc.
As well, the identity of the proteins encoded by the herein disclosed nucleic acid molecules will enable the rapid screening of a large number of compounds to identify those candidates suitable for further, in-depth studies of therapeutic applications.